Pathogenic infections represent a persistent threat to human health. The rapid development of resistance to drug therapies creates a continuing need for new anti-infective agents. The proposed research focuses on the development of sequence-random copolymers having a nylon-3 backbone as general, inexpensive antibacterial agents. We will synthesize and characterize a variety of these copolymers designed to mimic the activity of natural host-defense peptides (HDPs), which have been discovered in plants, animals, and humans. HDPs are remarkable for their general ability to halt growth of both Gram negative and Gram positive bacteria while leaving animal cells largely unaffected. The relative inability of bacteria to resist HDPs is usually attributed to a general mode of action involving degradation of bacterial cell membranes. Cationic HDPs are known to form globally amphipathic helices (hydrophobic side chains on one side, charged and other hydrophilic side chains on the opposite side) when they bind to anionic cell membranes. In contrast, the nylon-3 copolymers of interest contain a random sequence of cationic and hydrophobic side chains;they also vary in length. Preliminary work suggests that the random copolymers attack membranes by forming irregular amphipathic surface structures enabled by the flexibility of the nylon-3 backbone. Several of the random nylon-3 copolymers have bacteriostatic properties rivaling those of natural HDPs;they hemolytically attack red blood cells only at high concentration. This work will obtain a better fundamental understanding of how these copolymers degrade membranes in order to inform design improvements. The approach includes both chemical synthesis and detailed analysis of function by single-cell fluorescence imaging. The synthetic methodology enables control of mean copolymer length and the type and percentage of hydrophobic, cationic, and polar side chains. For each individual cell, the analytical methods enable direct correlation in real time of the development of bacterial "symptoms" with the amount of antimicrobial polymer absorbed and the halting of cell growth. The initial work will focus on E. coli and B. subtilis as representative Gram negative and Gram positive species. Observable symptoms include translocation across the outer membrane (OM), lysing of the OM, translocation across the cytoplasmic membrane (CM) and lysing of the CM. The same techniques will determine the special properties of "survivor cells" that are unusually resistant to attack. Time lapse observations after restoration of normal growth medium will reveal which short-term symptoms are sufficient to kill cells, i.e. to prevent subsequent recovery and growth. Detailed mechanistic data from the experiments will feed back into the effort to design cheap and effective antimicrobial polymers. The novel techniques and concepts developed in this work will find wide application in all efforts to design antimicrobial agents involving membrane-based functions. Random copolymers may find applications in the context of antifungal activity, antiplasmodial activity, and lung-surfactant activity as well. PUBLIC HEALTH RELEVANCE: Bacterial infections are increasingly resistant to antibiotic therapies. This work explores random copolymers based on the nylon-3 backbone as a new class of antibacterial agents. Fundamental insights from detailed experimental work will inform the design of more effective antibacterial polymers. These can be synthesized in large quantities at low cost.